肺炎克雷伯菌外膜蛋白A慢病毒载体的构建及其对Raw264.7细胞NOD样受体热蛋白结构域相关蛋白3炎症小体的激活

Construction of a lentiviral vector encoding outer membrane protein A from Klebsiella pneumoniae and its activation of NOD-like receptor thermal protein domain associated protein 3 inflammasome in Raw264.7 cells

目的探究肺炎克雷伯菌(KP)外膜蛋白A(OmpA)是否可以激活Raw264.7巨噬细胞NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体。方法根据(美国)国家生物技术信息中心(NCBI)公布的KP OmpA基因序列(GenBank Number:AJ000998.1),以本科室保存的KP菌株基因组为模板,设计引物,通过聚合酶链反应(PCR)扩增OmpA全长序列并经同源重组酶连接至慢病毒表达载体pLVX-AcGFP1-N1。将该重组表达载体与慢病毒包装质粒pLP1,pLP2和pLPVSV-G共转染HEK293T细胞,收集慢病毒液并感染Raw264.7巨噬细胞,经有限稀释法筛选获得表达KP OmpA的Raw264.7单克隆细胞株。使用蛋白质印迹法(Western blot)和酶联免疫吸附试验(ELISA)检测NLRP3、半胱氨酸蛋白酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)表达。组间比较采用t检验。结果成功包装出慢病毒并获得稳定表达KP OmpA的Raw264.7细胞系,KP OmpA稳转细胞系胞内NLRP3、Caspase-1、ASC、IL-1β灰度值显著高于空载细胞系(1.17±0.01比0.71±0.01,t=31.170,P<0.01;2.25±0.06比1.43±0.07,t=8.909,P<0.05;1.82±0.06比1.25±0.01,t=8.900,P<0.05;1.41±0.06比0.58±0.01,t=12.860,P<0.01)。KP OmpA稳转细胞系细胞上清中IL-1β和IL-18表达量显著高于空载细胞系[(215.70±14.93)pg/ml比(20.37±9.86)pg/ml,t=10.920,P<0.01;(115.90±14.88)pg/ml比(15.40±4.72)pg/ml,t=6.434,P<0.05]。结论肺炎克雷伯菌外膜蛋白OmpA可以激活Raw264.7巨噬细胞NLRP3炎症小体,并上调炎性因子IL-1β和IL-18表达。

Objective To explore whether Klebsiella pneumoniae(KP)outer membrane protein A(Outer membrane protein A,OmpA)activate NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome in Raw264.7 macrophage.Methods According to(US)National Center for Biotechnology Information(NCBI)the published KP OmpA gene sequence(GenBank Number:AJ000998.1),using the KP genome preserved in our department as a template,designed primers,using PCR amplified the fragment and connected it to the lentiviral expression vector pLVX-AcGFP1-N1 by homologous recombination.The recombinant expression vector was co-transfected with lentiviral packaging plasmids pLP1,pLP2 and pLPVSV-G into HEK293T cells,the lentiviral solution was collected and to infect Raw264.7 macrophages,the Raw264.7 monoclonal cell expressing KP OmpA was screened by limiting dilution method.The expressions of NLRP3,cysteinyl aspartate specific proteinase-1(Caspase-1),apoptosis-associated speck-like protein containing a CARD(ASC),interleukin-1β(IL-1β)and interleukin-18(IL-18)were detected by Western blotting and enzyme-linked immunosorbent assay(ELISA).T test was used to cOmpAre between groups.Results The lentivirus was successfully packaged and the Raw264.7 cell line stably expressing KP OmpA was obtained.The gray value of NLRP3,Caspase-1,ASC,and IL-1βin the KP OmpA cell line was significantly higher than that of the vector cell line(1.17±0.01 vs.0.71±0.01,t=31.170,P<0.01;2.25±0.06 vs.1.43±0.07,t=8.909,P<0.05;1.82±0.06 vs.1.25±0.01,t=8.900,P<0.05;1.41±0.06 vs.0.58±0.01,t=12.860,P<0.01).The expressions of IL-1βand IL-18 in the supernatant of the KP OmpA cell line were significantly higher than those in the vector cell line[(215.70±14.93)pg/ml vs.(20.37±9.86)pg/ml,t=10.920,P<0.01;(115.90±14.88)pg/ml vs.(15.40±4.72)pg/ml,t=6.434,P<0.05].Conclusion Klebsiella pneumoniae outer membrane protein OmpA can activate NLRP3 inflammasome in Raw264.7 macrophages,and up-regulate the expression of inflammatory factors IL-1βand IL-18.